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Protein mediated synthesis of Au–Ag bimetallic nanoparticles
Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Protein mediated synthesis of Au–Ag bimetallic nanoparticles D. Raju, Ritul Mendapara, Urmil J. Mehta n Plant Tissue Culture Division, CSIR – National Chemical Laboratory, Pune 411008, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 15 August 2013 Accepted 15 March 2014
There is growing need to develop environment friendly method for synthesis of bimetallic nanoparticle that do not use toxic chemicals during their synthesis process. Here, we report synthesis of Au–Ag bimetallic nanoparticles by using macerase enzyme as reducing agent at different temperatures. Bimetallic nanoparticles were synthesized at 80 and 90 1C. The intensity of formation of nanoparticles was more at 80 1C. The bimetallic nanoparticles are characterized by using UV–vis, TEM and EDAX. The TEM study shows the inner gold and outer silver bimetallic nanoparticles. The formed nanoparticles are 4–20 nm in size and spherical in shape. EDAX confirms the formed bimetallic nanoparticles are of gold and silver. & 2014 Published by Elsevier B.V.
Keywords: Synthesis Gold Silver Macerase Enzyme Particles
1. Introduction The developments in the field of nanotechnology in recent and past, with different methodologies to synthesize nanoparticles of particular shape and size are continuing. Nanoparticle synthesis by physical, chemical methods as laser ablation, pyrolysis, chemical vapor deposition, sol–gel technique and electro-deposition are studied. These processes of synthesis are expensive and hazardous [1,2]. Currently, there is a growing need to develop environment friendly methods for synthesizing nanoparticle where the synthesis processes do not involve toxic chemicals. As a result, researchers have turned to the field of nanoparticle synthesis by using biological systems. The role of microorganisms and plants in the synthesis of nanoparticles has been recently reviewed [3]. Biological methods of nanoparticles synthesis using microorganisms [4–6], enzymes [7], and plant extracts [8] have been suggested as possible eco-friendly alternatives to chemical and physical methods. Extracellular synthesis of Au, Ag and Au–Ag nanoparticles in water, using the extract of Volvariella volvacea, a naturally occurring edible mushroom, as reducing and protecting agents has been demonstrated [9]. The green chemistry approach for the biosynthesis of Au, Ag, Au–Ag alloy and Au core–Ag shell nanoparticles using the aqueous extract and dried powder of Anacardium occidentale leaf was studied. [10]. A simple and reproducible method for the synthesis of triangular Au core–Ag shell nanoparticles has been demonstrated [11]. The use of algal cells for the biosynthesis of pure metallic silver, gold and Au core/Ag shell nanoparticles by simultaneous reduction of aqueous AgNO3 and n
Corresponding author. Tel.: þ 91 20 25902217; fax: þ 91 20 25902645. E-mail address: [email protected] (U.J. Mehta).
HAuCl4 has also been carried out [12]. The synthesis of bimetallic Au/Ag nanoparticles by the competitive reduction of Au3 þ and Ag þ ions using Persimmon leaf broth was studied [13]. There are few reports on synthesis of bimetallic nanoparticles by different microorganisms and plant extracts. However, much research has not been carried out on bimetallic nanoparticles by using protein. Here we report the synthesis of bimetallic nanoparticles by using macerase enzyme at different temperatures.
2. Materials and methods Macerase enzyme preparation: Macerase enzyme was used as Q2 reducing agent for bimetallic nanoparticles (NPs). It was purchased from CALBIOCHEMs. An amount of 125 mg enzyme powder was added to 5 ml distilled water and used as stock solution. An amount of 200 ml enzyme was used for bimetallic NPs synthesis. Synthesis of Au–Ag bimetallic nanoparticles: The bimetallic gold– silver nanoparticles were prepared by adding both chloroauric acid (HAuCl4) and silver nitrate (AgNO3) solution of 0.1 mM concentration in equal ratio (1:1). UV analysis: The samples were scanned from 300–800 nm wavelengths with a UV spectrophotometer (SHIMANDZU UV2450) using a dual beam operated at 1 nm resolution. The reaction mixtures were also scanned under the same wavelengths after every 2 h upto a period of 12 h. Transmission electron microscopy: The morphology of bimetallic nanoparticle images was recorded with TEM for determination of the shape and size. Two to three drops of bimetallic nanoparticle solution were placed on carbon coated copper grids and allowed
http://dx.doi.org/10.1016/j.matlet.2014.03.087 0167-577X/& 2014 Published by Elsevier B.V.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i
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D. Raju et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 1. Visual observation of Ag–Au bimetallic nanoparticles at different temperatures (A) room temperature, (B) 60 1C, (C) 80 1C and (D) 90 1C. (For interpretation of the references to color in this figure the reader is referred to the web version of this article.)
Fig. 2. UV–visible spectrum for Ag–Au bimetallic nanoparticles synthesis at different temperature, (A) room temperature, (B) 60 1C, (C) 80 1C and (D) 90 1C.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i
D. Raju et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
to dry. TEM measurements were done on JOEL model 1200EX instrument operated at an accelerating voltage of 200 kV. The particles size distribution was calculated by using Gatan software.
3. Results and discussion Visual analysis: After the addition of 200 ml enzyme to the solution there was change in the color of the solution at different temperatures. The color of the solutions at room temperature (RT) and 60 1C was purple in color (Fig. 1A and B). It was brownish purple in color at temperatures 80 and 90 1C indicating formation of bimetallic nanoparticles (Fig. 1C and D). UV analysis: The biosynthesis of bimetallic gold and silver nanoparticles synthesized after addition of 200 ml of macerase enzyme was monitored by UV–vis spectra. The reaction was performed at different temperatures (RT, 60, 80 and 90 1C) and at different time intervals. The peak at 540 and 535 nm was observed at RT and 60 1C reaction that corresponds to gold nanoparticles
3
(Fig. 2A and B) thus indicating the formation of gold nanoparticles. In the reaction at 80 and 90 1C, at the initial stage of reaction at 2 and 4 h there was a peak at 530 nm that later shifted to 510 nm indicating the formation of bimetallic nanoparticles (Fig. 2C and D). In previous report also it was demonstrated that peak at 510 nm corresponds to bimetallic nanoparticles [12]. The shift in the peak over a time period could possibly be due to the formation of gold nanoparticles at the initial period; as the time increases the gold nanoparticles were covered with outer silver layer and the peak shifted to the lower wavelength. The spectra of RT and 60 1C reaction show an increase in the intensity of peak with time. However, in the reaction at 80 1C the increase in the intensity of peak was noted till 10 h which reduced at 12 h, while in the reaction at 90 1C the intensity of peak reduced gradually with time period. This reduction in the intensity of peak can be attributed to the time dependent loss of enzyme activity at higher temperature. The intensity of bimetallic NP formation was more in 80 1C than other reaction conditions, so we performed TEM analysis of 80 1C reaction.
Fig. 3. TEM images of Ag–Au bimetallic nanoparticles synthesized at 80 1C.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i
D. Raju et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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TEM analysis: The TEM images recorded were from the spherical Au–Ag bimetallic nanoparticles formed by the reduction of 0.1 M AgNO3 and 0.1 M HAuCl4 by macerase enzyme. The reduction of silver ions bound to the gold nanoparticles by macerase enzyme results in the formation bimetallic nanoparticles at 80 and 90 1C. We observed nanoparticles made up of inner gold core and outer silver shell. The bimetallic nanoparticles were of different sizes (Fig. 3A and B). In an earlier report the reduction of silver ions bound to the gold nanotriangles by ascorbate ions helped in formation of silver atoms and nucleated to grow on the surface of the gold nanotriangle [11]. The energy dispersive X-ray analysis (EDAX) shows Au–Ag bimetallic nanoparticles. EDAX data shows the peak of Au and Ag present in sample, which clearly indicates the formation of bimetallic nanoparticles (Fig. 3C). There is also a peak of Cu in the EDAX data, which comes from the Cu TEM grid. The size distribution of bimetallic nanoparticles was studied using Gatan software. The size was calculated by measuring hundred bimetallic nanoparticles. The nanoparticles formed are circular in shape and well separated with no agglomeration. Nanoparticle sizes were between 4 and 20 nm, the maximum number of nanoparticles being 9 nm in size (Fig. 3D). 4. Conclusions The macerase enzyme mediated synthesis of bimetallic nanoparticles of gold and silver has been demonstrated in the present report. The formed nanoparticles were small in size having potential application in medicine. The synthesis of nanoparticles was carried out at different temperatures where bimetallic
nanoparticles were formed at 80 and 90 1C. The intensity of formation of nanoparticles was more at 80 1C. This macerase enzyme mediated green approach towards the synthesis of Au– Ag bimetallic nanoparticles reduces or eliminates the use and generation of hazardous substances.
Acknowledgments Authors are grateful to the Council of Scientific and Industrial Q3 Research (CSIR), New Delhi for financial support. The authors also thank the Center for Material Characterization, NCL for TEM analysis. References [1] Ankamwar B, Chaudhary M, Sastry M. Synth React Inorg Met Org Chem 2005;35:19–26. [2] Roy N, Barik A. Int J Nanotechnol Appl 2010;4:95–101. [3] Mohanpuria P, Rana NK, Yadav SK. J Nanopart Res 2008;10:507–17. [4] Klaus T, Joerger R, Olsson E, Granqvist CG. Proc Natl Acad Sci USA 1999;96:13611–4. [5] Konishi Y, Ohno K, Saitoh N, Nomura T, Nagamine S, Hishida H, Takahashi Y, Uruga T. J Biotechnol 2007;128:48–53. [6] Nair B, Pradeep T. Cryst Growth Des 2002;2:293–8. [7] Willner I, Baron R, Willner B. Adv Mater 2006;18:1109–20. [8] Raju D, Hazra S, Mehta UJ. Mater Lett 2013;102–103:5–7. [9] Philip D. Spectrochim Acta Part A 2009;73:374–81. [10] Shenya DS, Mathewa J, Philip D. Spectrochim Acta Part A 2011;79:254–62. [11] Rai A, Chaudhary M, Ahmad A, Bhargava S, Sastry M. Mater Res Bull 2007;42:1212–20. [12] Govindaraju K, Khaleel S, Vijayakumar B, Kumar G, Singaravelu G. J Mater Sci 2008;43:5115–22. [13] Song JY, Kim BS. Korean J Chem Eng 2008;25:808–11.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Q1 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Q5 64 65 66
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Protein mediated synthesis of Au–Ag bimetallic nanoparticles D. Raju, Ritul Mendapara, Urmil J. Mehta n Plant Tissue Culture Division, CSIR – National Chemical Laboratory, Pune 411008, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 15 August 2013 Accepted 15 March 2014
There is growing need to develop environment friendly method for synthesis of bimetallic nanoparticle that do not use toxic chemicals during their synthesis process. Here, we report synthesis of Au–Ag bimetallic nanoparticles by using macerase enzyme as reducing agent at different temperatures. Bimetallic nanoparticles were synthesized at 80 and 90 1C. The intensity of formation of nanoparticles was more at 80 1C. The bimetallic nanoparticles are characterized by using UV–vis, TEM and EDAX. The TEM study shows the inner gold and outer silver bimetallic nanoparticles. The formed nanoparticles are 4–20 nm in size and spherical in shape. EDAX confirms the formed bimetallic nanoparticles are of gold and silver. & 2014 Published by Elsevier B.V.
Keywords: Synthesis Gold Silver Macerase Enzyme Particles
1. Introduction The developments in the field of nanotechnology in recent and past, with different methodologies to synthesize nanoparticles of particular shape and size are continuing. Nanoparticle synthesis by physical, chemical methods as laser ablation, pyrolysis, chemical vapor deposition, sol–gel technique and electro-deposition are studied. These processes of synthesis are expensive and hazardous [1,2]. Currently, there is a growing need to develop environment friendly methods for synthesizing nanoparticle where the synthesis processes do not involve toxic chemicals. As a result, researchers have turned to the field of nanoparticle synthesis by using biological systems. The role of microorganisms and plants in the synthesis of nanoparticles has been recently reviewed [3]. Biological methods of nanoparticles synthesis using microorganisms [4–6], enzymes [7], and plant extracts [8] have been suggested as possible eco-friendly alternatives to chemical and physical methods. Extracellular synthesis of Au, Ag and Au–Ag nanoparticles in water, using the extract of Volvariella volvacea, a naturally occurring edible mushroom, as reducing and protecting agents has been demonstrated [9]. The green chemistry approach for the biosynthesis of Au, Ag, Au–Ag alloy and Au core–Ag shell nanoparticles using the aqueous extract and dried powder of Anacardium occidentale leaf was studied. [10]. A simple and reproducible method for the synthesis of triangular Au core–Ag shell nanoparticles has been demonstrated [11]. The use of algal cells for the biosynthesis of pure metallic silver, gold and Au core/Ag shell nanoparticles by simultaneous reduction of aqueous AgNO3 and n
Corresponding author. Tel.: þ 91 20 25902217; fax: þ 91 20 25902645. E-mail address: [email protected] (U.J. Mehta).
HAuCl4 has also been carried out [12]. The synthesis of bimetallic Au/Ag nanoparticles by the competitive reduction of Au3 þ and Ag þ ions using Persimmon leaf broth was studied [13]. There are few reports on synthesis of bimetallic nanoparticles by different microorganisms and plant extracts. However, much research has not been carried out on bimetallic nanoparticles by using protein. Here we report the synthesis of bimetallic nanoparticles by using macerase enzyme at different temperatures.
2. Materials and methods Macerase enzyme preparation: Macerase enzyme was used as Q2 reducing agent for bimetallic nanoparticles (NPs). It was purchased from CALBIOCHEMs. An amount of 125 mg enzyme powder was added to 5 ml distilled water and used as stock solution. An amount of 200 ml enzyme was used for bimetallic NPs synthesis. Synthesis of Au–Ag bimetallic nanoparticles: The bimetallic gold– silver nanoparticles were prepared by adding both chloroauric acid (HAuCl4) and silver nitrate (AgNO3) solution of 0.1 mM concentration in equal ratio (1:1). UV analysis: The samples were scanned from 300–800 nm wavelengths with a UV spectrophotometer (SHIMANDZU UV2450) using a dual beam operated at 1 nm resolution. The reaction mixtures were also scanned under the same wavelengths after every 2 h upto a period of 12 h. Transmission electron microscopy: The morphology of bimetallic nanoparticle images was recorded with TEM for determination of the shape and size. Two to three drops of bimetallic nanoparticle solution were placed on carbon coated copper grids and allowed
http://dx.doi.org/10.1016/j.matlet.2014.03.087 0167-577X/& 2014 Published by Elsevier B.V.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i
2
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D. Raju et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 1. Visual observation of Ag–Au bimetallic nanoparticles at different temperatures (A) room temperature, (B) 60 1C, (C) 80 1C and (D) 90 1C. (For interpretation of the references to color in this figure the reader is referred to the web version of this article.)
Fig. 2. UV–visible spectrum for Ag–Au bimetallic nanoparticles synthesis at different temperature, (A) room temperature, (B) 60 1C, (C) 80 1C and (D) 90 1C.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i
D. Raju et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
to dry. TEM measurements were done on JOEL model 1200EX instrument operated at an accelerating voltage of 200 kV. The particles size distribution was calculated by using Gatan software.
3. Results and discussion Visual analysis: After the addition of 200 ml enzyme to the solution there was change in the color of the solution at different temperatures. The color of the solutions at room temperature (RT) and 60 1C was purple in color (Fig. 1A and B). It was brownish purple in color at temperatures 80 and 90 1C indicating formation of bimetallic nanoparticles (Fig. 1C and D). UV analysis: The biosynthesis of bimetallic gold and silver nanoparticles synthesized after addition of 200 ml of macerase enzyme was monitored by UV–vis spectra. The reaction was performed at different temperatures (RT, 60, 80 and 90 1C) and at different time intervals. The peak at 540 and 535 nm was observed at RT and 60 1C reaction that corresponds to gold nanoparticles
3
(Fig. 2A and B) thus indicating the formation of gold nanoparticles. In the reaction at 80 and 90 1C, at the initial stage of reaction at 2 and 4 h there was a peak at 530 nm that later shifted to 510 nm indicating the formation of bimetallic nanoparticles (Fig. 2C and D). In previous report also it was demonstrated that peak at 510 nm corresponds to bimetallic nanoparticles [12]. The shift in the peak over a time period could possibly be due to the formation of gold nanoparticles at the initial period; as the time increases the gold nanoparticles were covered with outer silver layer and the peak shifted to the lower wavelength. The spectra of RT and 60 1C reaction show an increase in the intensity of peak with time. However, in the reaction at 80 1C the increase in the intensity of peak was noted till 10 h which reduced at 12 h, while in the reaction at 90 1C the intensity of peak reduced gradually with time period. This reduction in the intensity of peak can be attributed to the time dependent loss of enzyme activity at higher temperature. The intensity of bimetallic NP formation was more in 80 1C than other reaction conditions, so we performed TEM analysis of 80 1C reaction.
Fig. 3. TEM images of Ag–Au bimetallic nanoparticles synthesized at 80 1C.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i
D. Raju et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
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TEM analysis: The TEM images recorded were from the spherical Au–Ag bimetallic nanoparticles formed by the reduction of 0.1 M AgNO3 and 0.1 M HAuCl4 by macerase enzyme. The reduction of silver ions bound to the gold nanoparticles by macerase enzyme results in the formation bimetallic nanoparticles at 80 and 90 1C. We observed nanoparticles made up of inner gold core and outer silver shell. The bimetallic nanoparticles were of different sizes (Fig. 3A and B). In an earlier report the reduction of silver ions bound to the gold nanotriangles by ascorbate ions helped in formation of silver atoms and nucleated to grow on the surface of the gold nanotriangle [11]. The energy dispersive X-ray analysis (EDAX) shows Au–Ag bimetallic nanoparticles. EDAX data shows the peak of Au and Ag present in sample, which clearly indicates the formation of bimetallic nanoparticles (Fig. 3C). There is also a peak of Cu in the EDAX data, which comes from the Cu TEM grid. The size distribution of bimetallic nanoparticles was studied using Gatan software. The size was calculated by measuring hundred bimetallic nanoparticles. The nanoparticles formed are circular in shape and well separated with no agglomeration. Nanoparticle sizes were between 4 and 20 nm, the maximum number of nanoparticles being 9 nm in size (Fig. 3D). 4. Conclusions The macerase enzyme mediated synthesis of bimetallic nanoparticles of gold and silver has been demonstrated in the present report. The formed nanoparticles were small in size having potential application in medicine. The synthesis of nanoparticles was carried out at different temperatures where bimetallic
nanoparticles were formed at 80 and 90 1C. The intensity of formation of nanoparticles was more at 80 1C. This macerase enzyme mediated green approach towards the synthesis of Au– Ag bimetallic nanoparticles reduces or eliminates the use and generation of hazardous substances.
Acknowledgments Authors are grateful to the Council of Scientific and Industrial Q3 Research (CSIR), New Delhi for financial support. The authors also thank the Center for Material Characterization, NCL for TEM analysis. References [1] Ankamwar B, Chaudhary M, Sastry M. Synth React Inorg Met Org Chem 2005;35:19–26. [2] Roy N, Barik A. Int J Nanotechnol Appl 2010;4:95–101. [3] Mohanpuria P, Rana NK, Yadav SK. J Nanopart Res 2008;10:507–17. [4] Klaus T, Joerger R, Olsson E, Granqvist CG. Proc Natl Acad Sci USA 1999;96:13611–4. [5] Konishi Y, Ohno K, Saitoh N, Nomura T, Nagamine S, Hishida H, Takahashi Y, Uruga T. J Biotechnol 2007;128:48–53. [6] Nair B, Pradeep T. Cryst Growth Des 2002;2:293–8. [7] Willner I, Baron R, Willner B. Adv Mater 2006;18:1109–20. [8] Raju D, Hazra S, Mehta UJ. Mater Lett 2013;102–103:5–7. [9] Philip D. Spectrochim Acta Part A 2009;73:374–81. [10] Shenya DS, Mathewa J, Philip D. Spectrochim Acta Part A 2011;79:254–62. [11] Rai A, Chaudhary M, Ahmad A, Bhargava S, Sastry M. Mater Res Bull 2007;42:1212–20. [12] Govindaraju K, Khaleel S, Vijayakumar B, Kumar G, Singaravelu G. J Mater Sci 2008;43:5115–22. [13] Song JY, Kim BS. Korean J Chem Eng 2008;25:808–11.
Please cite this article as: Raju D, et al. Protein mediated synthesis of Au–Ag bimetallic nanoparticles. Mater Lett (2014), http://dx.doi. org/10.1016/j.matlet.2014.03.087i